User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/08/05

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More product please...


  • As my final concentrations were so low, I decided to repeat the PCR of the other day, but now putting 4 reactions of the mutated luciferase and keeping the same controls.


--> Mix 1 for 30 μL <--

-H2O ----------------> 9μl
-Buffer 3.3x --------> 6μl
-Mg(OAc)2 ----------> 3μl
-dNTP's -------------> 5μl
-Primer Fw ----------> 3μl
-Suffix ---------------> 3μl
-DNA (3/50)---------> 1μl


--> Mix 2 for 20 μL <-- (this reaction will be added after the hot start)

-H2O ------------> 10.5μl
-Buffer 3.3 -----> 9μl
-rTth ------------> 0.5μl

  • The 35 cycles were programmed as follows:



- Initialization step: 94°C for 5 min. (only the 1st mix)

- Hot start: Stop to add the second mix

- Denaturation step: 94°C for 45 seg.

- Annealing step: 60°C for 45 seg.

- Extension/elongation step: 72°C for 3 min.

- Final elongation: 72°C for 10:00 min.

- Final hold: 4°C for ∞.

  • The gel shows that everything went ok :D





  • The pcr products were purified with Roche's® kit and stored as Mut-luz Prod PCR purif Mar and date under the number 2-O and 2-P


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